Brucella abortus internalization in HeLa cells 1
GTPases of the Rho subfamily are required for Brucella
abortus internalization in non-professional phagocytes: direct
activation of Cdc42.
Caterina Guzmán-Verri§§§§¶¶¶¶, Esteban Chaves-Olarte§§§§#, Christoph von
Eichel-Streiber‡‡, Ignacio López-Goñi‡, Monica Thelestam¶¶¶¶, Staffan
Arvidson¶¶¶¶, Jean-Pierre Gorvel†††† and Edgardo Moreno§§§§*
From the §§§§Programa de Investigación en Enfermedades Tropicales (PIET), Escuela de
Medicina Veterinaria, Universidad Nacional, Aptdo 304-3000 Heredia, Costa Rica ¶
Microbiology & Tumorbiology Center, Box 280, Karolinska Institute, S-17177
Stockholm, Sweden, #Centro de Investigación en Enfermedades Tropicales, Facultad de
Microbiología, Universidad de Costa Rica, 1000 San José, Costa Rica, ‡‡Institut für
Medizinische Mikrobiologie und Hygiene, Verfügungsgebaude für Forschung und
Entwicklung, Obere Zahlbacher Str.63, Johannes Gutenberg-Universität Mainz, 55101
Mainz, Federal Republic of Germany, ‡Departamento de Microbiología, Universidad
de Navarra, Aptdo 177, 31080, Pamplona, Spain and ††††Centre d’Immunologie INSERM-
CNRS de Marseille-Luminy, 13288 Marseille Cedex 9, France
Running title: B. abortus internalization in HeLa cells
*To whom correspondence should be addressed. Tel (506) 2380761 Fax (506) 2381298
e-mail: [email protected].
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on September 28, 2001 as Manuscript M105606200 by guest on M
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Brucella abortus internalization in HeLa cells 2
Members of the genus Brucella are intracellular alpha Proteobacteria responsible
of brucellosis, a chronic disease of humans and animals. Little is known about
Brucella virulence mechanisms, but the ability of these bacteria to invade and to
survive within cells are decisive factors for causing disease. Transmission electron
and fluorescence microscopy of infected non-professional phagocytes HeLa cells
revealed minor membrane changes accompanied by discrete recruitment of F-
actin at the site of Brucella abortus entry. Cell uptake of B. abortus was negatively
affected to various degrees by actin, actin-myosin and microtubule chemical
inhibitors. Modulators of mitogen-activated protein kinases and tyrosine protein
kinases hampered Brucella cell internalization. Inactivation of Rho small GTPases
using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522 and TcdA significantly
reduced the uptake of B. abortus by HeLa cells. On the contrary, cytotoxic
necrotizing factor from Escherichia coli, known to activate Rho, Rac and Cdc42
small GTPases, increased the internalization of both, virulent and non-virulent B.
abortus. Expression of dominant positive Rho, Rac, and Cdc42 forms in HeLa cells,
promoted the uptake of B. abortus, whereas expression of dominant negative forms
of these GTPases in HeLa cells, hampered Brucella uptake. Cdc42 was activated
upon cell contact by virulent B. abortus but not by a non-invasive isogenic strain,
as proven by affinity precipitation of active Rho, Rac and Cdc42. The polyphasic
approach used to discern the molecular events leading to Brucella internalization,
opens new alternatives for exploring the complexity of the signals required by
intracellular pathogens for cell invasion.
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Brucella abortus internalization in HeLa cells 3
Brucellosis is a contagious bacterial disease of animals and a true zoonosis. It is caused
by facultative intracellular organisms of the genus Brucella, composed by six
recognized species with affinity for different hosts (1-4). Infection in humans depends
upon contact with infected animals or their products, causing a severe syndrome which,
if left untreated, may lead to disability and death (4). Despite the fact that the first
member of the genus was described more than one hundred years ago, the intracellular
life cycle and virulence mechanisms of Brucella are just being unveiled (5-7). In
comparison with other pathogenic bacteria, Brucella lacks classical virulence factors
such as exotoxins, invasive proteases, toxic lipopolysaccharide, capsules, virulence
plasmids and lysogenic phages. Furthermore, it does not generate resistance forms, does
not display antigenic variation and lacks fimbria, pili and flagella (8). In general,
Brucella virulence resides in its well-developed ability to invade, survive and replicate
within vacuolar compartments of professional and non-professional phagocytes (6,9-
14). In professional phagocytes as well as in caprine M (lymphoepitelial) cells, Brucella
is ingested by a zipper-like mechanism (15). Opsonized brucellae are internalized via
complement and Fc receptors in macrophages and monocytes, whereas non-opsonized
Brucella seems to penetrate via lectin or fibronectin receptors, in addition to other
unknown receptors (16,17). In non-professional phagocytes, Brucella appears to be
internalized by receptor mediated phagocytosis (18,19). Although zipper-like
phagocytosis has been observed in these cells (7), it seems to be more an exceptional
event than a common phenomenon (18,20).
Penetration into non-professional phagocytes occurs within minutes after inoculation,
with one or two brucellae per cell (6). Cytoskeleton rearrangements have not been
directly observed but these structures seem to be required, since various cytoskeleton
chemical modulators hamper the internalization of Brucella in these cells (7,19).
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Brucella abortus internalization in HeLa cells 4
Although the molecular mechanisms underlying these phenomena are not known, at
least one signaling system, BvrR-BvrS, coding for a regulator (BvrR) and a sensor
protein (BvrS) is implicated in the invasion of B. abortus to cells (14). In the same
direction, the absence of O- and native hapten polysaccharides on Brucella surface
considerably varies bacterial cell invasion (14,17,21). These type of mutations are
known to modify the topology and biological properties of the Brucella outer
membrane, altering the attachment and penetration to host cells (22-24).
The ability of different bacteria to exploit cell signal transduction pathways and
cytoskeletal components to secure their survival is a well-recognized event. Paradigms
of host subversion by either intracellular or extracellular bacteria like Salmonella,
Shigella, Listeria, Neisseria, Yersinia and Escherichia have been established in recent
years (25-31). By interacting with cytoskeletal regulators, such as the small GTP-
binding proteins of the Rho subfamily, these bacteria have developed efficient ways to
induce cytoskeletal rearrangements. GTPases of the Rho subfamily function as
molecular switches that cycle between an active GTP bound state and an inactive GDP
bound state. Activated proteins of the Rho subfamily interact with effector molecules to
produce biological responses involving actin reorganization. Some of these responses
involve membrane rearrangements implicated in several functions, one of them being
phagocytosis (32).
To characterize the basic molecular events that proceed after B. abortus binds to non-
professional phagocytic HeLa cells, several microscopical and biological strategies were
followed. Initially, we have employed cytoskeletal chemical modulators in cells
previous to infection. Then, we used bacterial toxins capable of modifying small
GTPases of the Rho family, as well as expression of dominant positive or negative
GTPase forms in cells during bacterial infection. Finally, we performed direct
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Brucella abortus internalization in HeLa cells 5
quantification of activated small GTPases after infecting with B. abortus. The data
obtained indicate that B. abortus modulates the host cell cytoskeleton in order to induce
its internalization.
EXPERIMENTAL PROCEDURES
Bacterial strains and plasmids. All strains were routinely grown in tryptic soy or Luria
Bertani medium. B. abortus 2308 NaIr is a wild type, virulent smooth-
lipopolysaccharide strain described elsewhere (33). B. abortus 2.13 is a smooth
lipopolysaccharide, non-invasive 2308 NaIr derivative with a Tn5 insertion in bvrS (14).
Salmonella typhimurium SL1344 (34) was obtained from Stéphane Méresse from
Centre d’Immunologie de Marseille-Luminy, France. E. coli expressing CNF1, plasmids
encoding Myc epitope tagged Cdc42V12 and Cdc42N17 derived from pMT90 (Philipe
Chavrier, Institut Curie-Section Recherche, Paris, France) and plasmids expressing Myc
epitope tagged RhoAV14, RhoAN19, Rac1V12, Rac1N17 derived from pEXV (35,36)
were provided by Gilles Flatau and Patrice Boquet from Institut Nacional de la Santé et
de la Recherche Médicale, Nice, France. TRBD, glutatione transferase tagged, was
expressed from plasmid pGEX-2T-TRBD and provided by Xiang-Dong Ren and Martin
Alexander Schwartz from The Scripps Research Institute, California, USA (37). PDB,
glutatione transferase tagged, was expressed from a derivative pGEX-2T plasmid and
obtained from Gary M. Bokoch from The Scripps Research Institute, California, USA
(38).
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Brucella abortus internalization in HeLa cells 6
Cell culture, microinjection and transfection. Cells were grown in Eagle’s minimal
essential medium supplemented with 5 % fetal bovine serum, 2.5 % sodium bicarbonate
and 1 % glutamine. Penicillin (100 units/ml) and streptomycin (100 µg/ml) routinely
added, were excluded from cell cultures during Brucella infections. For cell
microinjection, 5 × 105 HeLa cells were seeded on 13 mm glass slides and incubated for
24 h at 37oC in 5 % CO2. Cells were microinjected (FemtoJet®, Eppendorf) in the
nucleus with the selected plasmids at a concentration of 1 µg/ml in sterile distilled water
and infected with B. abortus as described below. After 16 h incubation in the presence
of 5 µg/ml gentamicin, cells were processed for immunofluorescence. Successfully
injected cells and intracellular bacteria were localized by immunofluorescence using an
anti-Myc antibody (clone 9E-10, Santa Cruz), a TRITC-conjugated anti-mouse antibody
(Sigma) and bovine FITC-conjugated anti-Brucella antibody (39). Cell transfection, was
carried out in 24-well tissue culture plates using Lipofectin and according to
manufacture’s instructions (GIBCO BRL). Brucella cell infections were performed as
described below.
Binding and invasion assays. HeLa cells were grown to subconfluency in 24-well tissue
culture plates at 37ºC under 5% CO2. Chemical cytoskeletal modulators (Sigma) listed
in Table I, were present through the experiments and used at concentration and
incubation times according to Rosenshine et al. (40). The chemical 2,3 butanedione
monoxime was used at a concentration of 7 nM for 30 min (41), PD098059 was used at
a concentration of 50 µM for 40 min (42) and wortmannin at a concentration of 50 nM
for 30 min (43). Clostridial TcdB-10463, TcdB-1470, TcdA and TcsL-1522 selective
toxin inhibitors of small GTPases were prepared as described (44). E. coli CNF was
purified according to Falzano et al. (45). Unless otherwise stated, toxins working
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Brucella abortus internalization in HeLa cells 7
concentration and incubation times used were as follows: 50 ng/ml of TcdB-10463 for
40 min; 50 ng/ml of TcdB-1470 for 40 min; 5 ng/ml of TcdA, overnight; 1µg/ml of
TcsL-1522, overnight and 3 ng/ml CNF for 2 h. Intoxication of HeLa cells was always
carried out prior B. abortus infection. After intoxication, the monolayer was washed
once with cold phosphate-buffered saline (0.01 M, pH 7.4) and kept at 4 ºC until
infection. Infections were carried out using an overnight culture of B. abortus diluted in
Eagle’s minimal essential medium to reach a concentration of 2.5×108 CFU/ml. The
inoculum was then added to the monolayer at a multiplicity of infection of 500 CFU/ml.
For Salmonella control experiments, the multiplicity of infection was 50 CFU/ml. Plates
were centrifuged at 300 x g at 4 ºC, incubated for 30 min at 37 ºC under 5 % CO2, and
washed 3 times with phosphate-buffered saline. Extracellular bacteria were killed by
adding Eagle’s minimal essential medium supplemented with 100 µg/ml gentamicin for
1 h at 37ºC under 5 % CO2. Plates were then washed with phosphate-buffered saline.
HeLa cells were lysed by adding 0.1 % Triton X-100 for 10 min. The samples were
collected, spined and resuspended in 110 µl of tryptic soy broth. Aliquots were plated in
tryptic soy agar and incubated at 37 ºC for 3 days for determination of CFU.
Immunofluorescence and transmission electron microscopy. For immunofluorescence
analysis, HeLa cells (5 × 105) were seeded on 13 mm glass slides, incubated until
subconfluency at 37 oC under 5 % CO2 and inoculated with bacteria as described above.
After five washing steps with phosphate-buffered saline, cells were fixed with ice cold 3
% paraformaldehyde (Merck) for 15 min. Samples were washed once and incubated for
10 min with 50 mM NH4Cl-phosphate buffered saline. Intracellular and extracellular
bacteria were detected and counted as previously described (11). Briefly, extracellular
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Brucella abortus internalization in HeLa cells 8
bacteria were labeled by using FITC-conjugated anti-Brucella antibody diluted 1/250
(in 10 % horse serum in phosphate-buffered saline), followed by washing steps.
Intracellular bacteria were detected by incubating the slides for 30 min with an anti-B.
abortus rabbit antiserum (39) diluted 1/250 in 10 % horse serum, containing 0.1 %
saponine (permeabilization step). Then cells were washed three times with 0.2 %
Tween-20 and incubated 30 min with a TRITC-conjugated anti-rabbit antibody
(Jackson ImmunoResearch Laboratories, Inc.), diluted 1/150 in 10 % horse serum and
0.1 % saponine. When needed, FITC-phalloidin (Sigma) was added at this point. Slides
were mounted in Mowiol solution and analyzed by phase contrast or fluorescence
microscopy. Counts of intracellular and extracellular bacteria were performed in at least
100 infected cells and were expressed as a mean and standard deviation of bacteria/cell.
The percentage of cells with associated bacteria was expressed as the mean and standard
deviation of cells with bound bacteria in five different 40 × fields. Statistical analysis
was performed using the Student’s t-test. For transmission electron microscopy, HeLa
monolayers infected with an overnight culture of B. abortus 2308NaIr were fixed with
2.5 % glutaraldehyde, 2 % paraformaldehyde in 0.1 M phosphate buffer. Samples were
placed in 1 % OsO4 solution for 1 h for postfixation, dehydrated in graded concentration
of ethanol and infiltrated with Spurr resin. Thin sections on 300 mesh colloidon-coated
grids were stained with uranyl acetate and lead Sato’s solution (46). Preparations were
examined with a Hitachi H-7100 electron microscope operating at 75 kV.
Quantification of GTP-Rho, GTP-Rac and GTP-Cdc42. For precipitation steps,
glutatione transferase tagged-TRBD and glutatione transferase tagged-PBD were
purified from cell lysates of E. coli strains harboring plasmids pGEX-2T-TRBD or
pGEX-2T-PBD, respectively and according to Ren et al. and Bernard et al. (37,38).
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Brucella abortus internalization in HeLa cells 9
HeLa cells grown in 6-well plates were infected for different time intervals with B.
abortus at a multiplicity of infection of 5000 CFU/cell. After incubation, cells were
washed with ice cold phosphate buffer saline and lysed with 500 µl ice cold
precipitation buffer (1% Triton X-100, 0.1 % SDS, 0.3 % Nonidet P40, 500 mM NaCl,
10 mM MgCl2, 50 mM Tris, pH 7.2). Lysates were clarified by centrifugation at 14000
rpm for 1 min. Twenty µl of lysate were saved as control of total GTPase content. GTP-
loaded Rho GTPases were precipitated with sepharose beads coupled to either
glutatione transferase-PBD or glutatione transferase-TRBD proteins. Samples were
incubated for 30 min at 4 ºC with shaking, washed with precipitation buffer and
resuspended in 25 µl sample buffer for SDS-PAGE analysis (47). Samples transferred to
a polyvinylidene difluoride membrane (Roche Molecular Biochemicals) were tested
with either rabbit antibodies against Rho or Cdc42 (Santa Cruz) or with an anti-Rac
monoclonal antibody (Transduction Laboratories). Probing and developing were
performed with peroxidase-labelled secondary antibodies and with a
chemiluminescence Western blotting kit (Pierce SuperSignal West Dura), respectively.
Cdc42-GTP, Rho-GTP and Rac-GTP levels were calculated by using Scion Image for
Windows as compared to control total Cdc42, Rho and Rac.
RESULTS.
Host cell cytoskeleton responds to B. abortus contact. To assess the role of the host
cell cytoskeleton in Brucella internalization, HeLa cells were infected with bacteria and
analyzed by transmission electron microscopy and immunofluorescence microscopy. In
agreement with previous investigations (11,48), few cells in a monolayer had associated
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Brucella abortus internalization in HeLa cells 10
bacteria (see below). At 30 min infection, bacteria were mostly located in cell to cell
contacts rather than in the cell body (see below). Minor host-cell membrane projections
were observed upon contact with bacteria (Fig. 1A). Under these experimental
conditions, zipper-like phagocytosis was not observed, despite that a considerable
number of intracellular Brucella were already found within vacuoles, as previously
reported (6). When infected cells were stained with FITC-phalloidin, a discrete
rearrangement of the actin cytoskeleton was observed at the site of contact between
Brucella and its host cell (Fig. 1B-D). To further identify eukaryotic components
required for B. abortus uptake, HeLa cells were treated with different cytoskeletal and
signal transduction modulators before infection with B. abortus. Inhibition of the
eukaryotic microtubule network with colchicine or nocodazole reduced Brucella
internalization to 40 % and 10 % respectively as compared to non-intoxicated cells (Fig.
2). Treatment of cells with drugs affecting the actin cytoskeleton also impared
internalization. Particularly, cytochalasin D almost abrogated Brucella uptake. These
results were in agreement with the observations made by electron and fluorescence
microscopy, indicating participation of the host actin cytoskeleton in Brucella uptake.
When tyrosine kinases inhibitors such as tyrphostin and genistein were used, the
percentage of internalized bacteria was reduced to 10 % and 20 % respectively as
compared to non-treated cells. Pretreatment of HeLa cells with the mitogen-activated
protein kinase kinases inhibitor PD098059 resulted in 50 % decrease in bacterial
invasion whereas pretreatment with the phosphatidylinositol 3-kinase inhibitor
wortmannin, reduced Brucella internalization to 10 %. Salmonella typhimurim SL1344
was included as a control of our test system. Fig. 2 demonstrates that the effects induced
by the various chemicals modulators were similar to those reported for Salmonella
elsewhere (Table I).
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Brucella abortus internalization in HeLa cells 11
B. abortus internalization is affected by modulation of GTPases activity by
bacterial toxins. Clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522 and TcdA
have been described as glucosyltransferases targeting different members of the Rho and
Ras subfamilies of small GTPases (49,50). They efficiently block the interaction of Rho
and Ras protein subfamilies with their effectors, leading to functionally inactive
GTPases (51). On the other hand, CNF from E. coli exerts the opposite effect, i.e
activation of Rho GTPases (52,53). Since these toxins are very specific for different
small GTPases involved in cytoskeleton functions such as membrane ruffling,
lamellipodia and stress fiber formation (51,54) they can be used to study the role of Rho
proteins in the internalization of different pathogens (55,56). HeLa cells treated for 40
min with TcdB-10463, TcdB-1470, or overnight with TcdA and TcsL-1522 exhibited
decreased Brucella internalization as compared to non-treated cells (Fig. 3A). In
contrast, when cells were treated with CNF for 2 h an approximate 10 fold increase in
internalization was obtained as compared to untreated cells (Fig. 3B). From these
experiments it was concluded that some of the toxin targets outlined in Fig. 3 are
relevant for Brucella uptake. Because Rho proteins are implicated in the regulation of
the actin cytoskeleton, it was important to know whether the observed inhibitory effect
was due to the direct action of the toxins on Rho proteins or to a secondary effect
inducing actin depolymerization. HeLa cells were then treated with a constant dose of
toxin for different time periods and infected with B. abortus. A marked reduction in
Brucella uptake was seen already after 15 min intoxication with TcdB-10463 and TcdB-
1470 as compared to non-treated cells (Fig. 4A). Since cytopathic effect was not evident
until 30-45min intoxication, it was concluded that the reduced internalization of
Brucella was not caused by secondary actin depolymerization. With CNF, increased
internalization was observed after 1 h treatment, with a peak at 2-3 hours. Membrane
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Brucella abortus internalization in HeLa cells 12
ruffling was evident after 2 h treatment (Fig. 4B). The percentage of internalization
dramatically decreased after 3 h, probably due to secondary effects such as
unavailability of free actin monomers.
CNF but not TcdB cell intoxication affects adhesion of B. abortus. Successful
bacterial invasion depends on two consecutive steps: binding and internalization (57).
Inhibition or promotion of B. abortus uptake in toxin treated cells as compared to non-
intoxicated cells may be due to altered binding and/or internalization. To distinguish
between these possibilities, double immunofluorescence to resolve intracellular from
extracellular bacteria in cells treated with TcdB-10463 and CNF was performed, and
counts compared to infected non-intoxicated cells (Fig. 5). Binding was not affected by
intoxication with TcdB-10463 for 15 min, since the mean number of bacteria per cell
was not significantly different between non-intoxicated and intoxicated cells (p>0.05).
However, the proportion of extracellular to intracellular bacteria was higher in treated
cells (p<0.05, Fig. 5A, graph a). At 40 min intoxication, 100 % of the cells exhibited
some degree of typical arborizing cytopathic effect induced by this toxin (Fig. 5B, panel
b, TcdB-10463) as described previously (58). Under these conditions, bacteria were
found mainly at the edges of cell body whereas in control cells, they were found in cell
to cell contacts (Fig. 5B, panels a-c, control). Since body retraction is more evident in
these intoxicated cells, it was easier to observe the preferential binding of bacteria to the
remaining of cell to cell contacts. After 40 min intoxication with TcdB-10463, the mean
number of bacteria/cell was not significantly different (p>0.05) from that of control
cells (Fig. 5A, graph a) and the proportion of extracellular bacteria was even higher than
in cells intoxicated for 15 min. It has been reported that the percentage of B. abortus
infected cells in HeLa cells monolayers is less than 50 % (11,48). We therefore
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Brucella abortus internalization in HeLa cells 13
analyzed if in intoxicated HeLa cells this percentage was somehow modified. Fig. 5A,
graph c shows that the percentage of cells with associated bacteria in TcdB-10463
treated monolayers was lower than in non-intoxicated monolayers. In our hands, the
percentage ranged between 10 % and 20 % in infected non-intoxicated cells and was 6.5
and 3.6 % in TcdB-10463 treated monolayers for 15 and 40 min respectively, showing
that toxin treatment decreases infection. Altogether these results indicate that binding of
B. abortus to HeLa cells is not significantly affected by TcdB-10463 intoxication.
However, internalization is reduced because less bacteria were taken up per cell and less
cells in the monolayer had associated bacteria. Similar experiments were performed in
CNF treated HeLa cells. Membrane ruffling was recorded after 2 h intoxication and
bacteria were observed on the cell body (Fig. 5B, panels a-c, CNF), particularly close to
ruffles. Electron transmission microscopy of CNF treated HeLa cells infected with
Brucella indicated that bacteria are able to penetrate through membrane ruffles, when
present (not shown). Adhesion of virulent B. abortus 2308 to HeLa cells was promoted
by CNF treatment, as compared to non-treated cells (p<0.05, Fig. 5A, graph b).
However, the proportion of intracellular and extracellular bacteria did not differ
between control and intoxicated cells (p>0.05). The increased binding was not specific
for the virulent strain, because the internalization deficient strain, 2.13 (14) also bound
more to CNF treated cells than to non-treated cells (p<0.05). With strain 2.13 however,
the ratio of intracellular/extracellular bacteria was increased, because more bacteria
were found intracellularly (Fig 5A, graph b). Therefore, CNF intoxicated HeLa cells
promoted both, binding and internalization of the non-virulent strain 2.13. With virulent
strain 2308, no difference in the ratio of intracellular/extracellular bacteria was observed
after 30 min incubation, despite the fact that binding was promoted. On the other hand,
the percentage of cells associated with bacteria was significantly higher (p<0.01) in
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Brucella abortus internalization in HeLa cells 14
CNF treated cells for both, the virulent and non-virulent B. abortus strains (Fig. 5A,
graph d). In conclusion, CNF treatment of HeLa cells promotes Brucella binding per
cell and increases the number of cells with associated bacteria, leading to an overall
more efficient invasion of the cell monolayer.
B. abortus internalization is affected by the expression of dominant positive or
negative Rho GTPases. To further investigate the role of small GTPases in Brucella
uptake, infections of HeLa cells expressing active forms of Rho, Rac and Cdc42 were
performed. HeLa cells were microinjected with plasmids encoding Myc-tagged
dominant positive mutants of Rho, Rac and Cdc42. B. abortus 2308 was incubated for
30 min followed by addition of gentamicin to kill extracellular bacteria. After 16 h
gentamicin incubation, when bacterial replication is still not evident in control cells (6),
infected monolayers were processed for immunofluorescence. Expression of the
corresponding mutant Rho protein was verified by using immunofluorescence labeled
anti-Myc antibodies as shown in Fig. 6A. The number of intracellular bacteria/cell
increased in cells expressing positive mutant Rac and Rho but not Cdc42, as compared
to control cells (Fig. 6B, graph a). However, the percentage of cells with internalized
bacteria increased in all cases (Fig. 6B, graph b). As expected, the expression of
dominant negative Rho protein mutants, RhoAN19, Rac1N17 and Cdc42N17 in
transfected HeLa cells, inhibited to different extents the internalization of this bacterium
(Fig. 7), supporting a role for these small GTPases in Brucella uptake.
Cdc42 is directly activated by virulent but not by non-virulent B. abortus The
experiments described above indicated that active Rho, Rac and Cdc42 promote
Brucella uptake by HeLa cells. However, it was important to establish if binding of B.
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Brucella abortus internalization in HeLa cells 15
abortus to HeLa cells leads to direct activation of any of the Rho proteins. Lysates from
cells infected with either the virulent 2308 or non-invasive 2.13 strain were incubated
with beads bearing the Rho effector TRBD or the Rac and Cdc42 effector PBD,
according to the affinity capture systems developed by Ren et al. (37) and Bernard et al.
(38), respectively. After protein elution, samples were analyzed by Western Blot using
anti-RhoA, anti-Rac or anti-Cdc42 antibodies. Fig. 8A shows that no difference in Rho
or Rac activation was detected up to 60 min infection with the virulent 2308 strain. On
the contrary, increased levels of GTP-Cdc42 up to four fold, were detected at 30 min
after infection (Fig. 8B). Cdc42 activation was specific for the virulent strain, since the
internalization deficient 2.13 strain did not activate Cdc42 up to 60 min after infection.
It is therefore concluded that early direct Cdc42 activation is biologically important for
successful B. abortus internalization.
DISCUSSION
Different attempts have been made to characterize the host-parasite interactions that
prevail during Brucella entry into eukaryotic cells. Pathological and microscopic studies
have been reported (15,18,59,60), but the molecular mechanisms involved in the
process have not properly addressed. Evident membrane rearrangements have been
described upon Brucella infection of caprine M (limphoepithelial) cells and
macrophages (15,20). Our electron microscopy studies confirmed the results obtained
earlier (7,18), where only slight membrane rearrangements were found at the site of
virulent smooth Brucella entry in non-professional phagocytes. Moreover, phalloidin
staining demonstrated a modest recruitment of F-actin cytoskeleton at the site of the
attachment. The participation of actin cytoskeleton was further indicated by reduced
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Brucella abortus internalization in HeLa cells 16
internalization of Brucella after treatment of HeLa cells with the actin depolymerizing
agent cytochalasin D or with the myosin inhibitor 2,3 butanedione monoxime. Although
less dramatic than cytochalasin D, microtubule depolymerizing agents, also hampered
the invasion of Brucella to cells. Other investigators have arrived to similar conclusions
by using cytoskeletal inhibitors (7,19). However, it must be pointed out that this
inhibition could be the result of the indirect microtubule inhibitors effect on the MAP
kinase pathway (61-64), which is required for Brucella internalization as shown here.
Uptake of different bacteria depend on the actin cytoskeleton (65-75). Although
examples of bacteria requiring only the microtubule network for successful
internalization are rare (76), there are many bacteria that recruit both, microtubules and
microfilaments (77-84). In this respect, B. abortus appears to belong to this last group.
Given the growing evidence for potential interactions between the microtubule and actin
networks, it is feasible that pathogens exploiting one network would also be dependent
on the other (85-87). Involvement of host kinases, particularly tyrosine protein kinases
in Brucella internalization was suggested by the reduced internalization of bacteria by
HeLa cells intoxicated with two tyrosine protein kinases specific drugs, such as
tyrphostin and genistein. Furthermore, according to the results obtained with PD098059
intoxicated cells, the extracellular-signal-regulated kinase pathway also appears to be
required for Brucella uptake to some extent, indicating that Brucella is able to trigger a
response in its host cell upon contact. Phosphatidylinositols are also involved in this
process, as suggested by the decreased entry of B. abortus in cells pretreated with
wortmannin. Phosphatidylinositol 3-kinase has been pointed as both an upstream and
downstream effector of small GTPases (88-90) affecting actin polymerization that
eventually could lead to a GTPase dependent Brucella internalization event. A
converging molecule for all the pathways herein studied is Ras, a small GTPase
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activated upon ligand binding to its membrane receptor, particularly tyrosine kinase
receptors, coupling intracellular signal transduction pathways to changes in the external
environment. There is enough evidence to point the Raf-MEK-mitogen activated protein
kinase pathway as a key effector in Ras signaling (54). On the other hand,
phosphatidylinositol 3-kinase, can bind to GTP-Ras (91) and there is evidence that Ras
and Rho GTPases interact and are activated in series (32). It would be then relevant to
test if Ras is needed for Brucella invasion. According to the results obtained with the
chemical drugs, this transductional pathway could be similar to the one exploited by
Listeria, which appears to be different from the one used by Salmonella (Table I). This
idea is in agreement with the slight actin recruitment induced by Listeria and Brucella
but not by Salmonella, which induces a major recruitment (26,67,69).
Gentamicin survival assays using bacterial toxins treated cells demonstrated that Rho,
Rac and Cdc42 are needed for efficient Brucella internalization. This is also supported
by the reduction in bacteria entry in cells expressing dominant negative mutants of Rho,
Rac and Cdc42 GTPases. Cdc42, but not Rac or Rho was directly activated upon B.
abortus contact with host cells, an event exclusively observed with the virulent strain.
Since some clostridial toxins affecting Brucella invasion do not use Cdc42 as substrate,
it is feasible to conclude from these experiments the participation of other GTPases. In
this sense, it is possible that Brucella does not directly activate Rho and Rac, as well as
other Ras proteins, but takes advantage of activated GTPase pools kept in cells during
normal conditions. The increase in B. abortus uptake observed after cell treatment with
CNF, and the significant increase observed in cells microinjected with positive forms of
Rac and Rho, support this asseveration. Nevertheless, other GTPases such as Ral and
Rap, implicated in endocytosis (92-94), could be involved in the internalization process
as well.
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It is important to point out that both, TcdB-10463 and TcdB-1470 use the same cell
receptor and display very similar enzymatic parameters during cell intoxication. These
two toxins, however, differ in their substrate preference (49): while TcdB-10463
modifies Rho, Rac and Cdc42, TcdB-1470 uses Rac, as the only member of the Rho
subfamily. B. abortus internalization is affected earlier by TcdB-10463 than by TcdB-
1470 intoxication as shown by the time curves performed with these two toxins.
Whereas this observation supports the participation of the three GTPases from the Rho
subfamily during B. abortus internalization, the B. abortus almost 100% inhibition by
TcdB-1470 at later times reflects the importance of Rac. Indeed, Rac has recently been
described as a potential link between the microtubule and actin networks, since
microtubule growth induces Rac activation and therefore lamellipodia formation (87).
The results obtained by performing intoxication time curves proof that not only the
toxin kinetics but also the small GTPases physiology should be taken into account when
using this kind of tools. Once bound to their target, the toxins block Rho GTPases in
either a GTP or GDP bound state. In each of these states, these GTPases have different
downstream effects that are time dependent. It is important to evaluate the intoxication
output at early times, when is more likely to observe the direct effect of the toxins in
their Rho targets than downstream effects of the small GTPase intoxicated state. This is
clearly exemplified by CNF treated cells for periods longer than 3 hours (Fig. 4B).
Binding of B. abortus to HeLa cells was not affected by TcdB-10463 treatment for 15
or 40 min. However, according to the gentamicin survival assay, TcdB-10463 treatment
for 40 min affected B. abortus uptake. Double immunofluorescence experiments
indicated that bacteria were binding to cells but less number were internalized and less
number of cells had associated bacteria, explaining this phenomenon. CNF cell
intoxication affected Brucella invasion in different aspects: i) increased binding of
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Brucella abortus internalization in HeLa cells 19
bacteria/cell with an absolute increase of intracellular bacteria, ii) increased
internalization in the case of the B. abortus 2.13 mutant strain, with more intracellular
bacteria than in control experiments and iii) increased percentage of cells permissive to
B. abortus internalization. The 10 fold increase in internalization observed in the
gentamicin survival assay, should be the sum of these events, where probably the
augmented number of infected cells has a major contribution. This permissibility event
is affected by toxin treatment, suggesting that GTPases of the Rho subfamily might
have either a direct or indirect role perhaps by controlling the formation of cell to cell
contacts where B. abortus binds or by regulating the expression of a protein particularly
found in these regions and required for bacteria to bind. More studies are needed to
clarify why bacteria are mainly found in cell to cell contacts and why some cells in the
same monolayer are more permissive to B. abortus invasion than others, an event also
described for Campylobacter jejuni and Listeria (95,96)
B. abortus cell uptake may induce a particular signal transduction pathway where small
GTPases are activated in series. Indeed, Ras has been reported as a Cdc42 activator, and
Cdc42 itself has been described as a Rac activator, while Rac activates or inhibits Rho
to varying degrees (88,97,98). Although the events leading to Brucella internalization
may follow a similar GTPase activation pathway, this may be a simple view of a more
intricate set of signals occurring during the invasion of intracellular pathogens to cells.
Acknowledgments - The authors thank Enrique Freer and Maribelle Vargas from the Electron
Microscopy Unit at the University of Costa Rica for their help with the electron transmission
microscopy studies and Daphnne Garita for her technical assistance.
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FOOTNOTES
• Caterina Guzmán-Verri was a recipient of a grant from the Swedish International
Development Agency (Sida-SAREC), as part of the Karolinska International
Research Training program. This work was partially supported by Research
contract ICA4-CT-1999-10001 from the European Community, RTD project
NOVELTARGETVACCINES, Ministerio de Ciencia y Tecnología/Consejo
Nacional de Ciencia y Tecnología, Costa Rica, Vicerrectoría de Investigación from
Universidad de Costa Rica, American Society for Microbiology MIRCEN award
and Ministerio de Ciencia y Tecnología, Spain (AGL2000-0305-C02-01).
1The abbreviations used are: CNF; cytotoxic necrotizing factor from E. coli; TRBD,
Rhotekin Rho binding domain; PBD, GTPase-binding domain of p21 activated kinase
1; TRITC, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate;
TcdB, Clostridium difficile toxin B; TcdA, C. difficile toxin A; TcsL, C. sordellii lethal
toxin; CFU, colony forming units; PAGE, polyacrylamide gel electrophoresis
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Brucella abortus internalization in HeLa cells 29
FIGURE LEGENDS.
FIG. 1. B. abortus induces minor cytoskeletal rearrangements in HeLa cells. A,
transmission electron microscopy of B. abortus entry into HeLa cells reveals discrete
cellular projections at the site of contact between cell and bacterium (black arrow). Bar
in frame corresponds to 0.4 µm. B-D, double immunofluorescence analysis of F-actin
and extracelullar B. abortus bound to HeLa cells. B, the arrow points to B. abortus
immunolabeled with rabbit anti-Brucella serum and TRITC-conjugated anti-rabbit IgG
after cell infection. C, the white arrow points to foci of actin polymerization stained
with FITC-phalloidin. D, superimposition of images B and C demonstrates co-
localization of B. abortus and actin rearrangement.
FIG. 2. B. abortus internalization is impaired by using chemical cytoskeletal
modulators. HeLa cells were treated with different chemical drugs and then infected
with B. abortus (black bars) or S. typhimurium (white bars). The effect in bacteria
uptake was assessed by using the gentamicin survival assay as described under
Experimental Procedures. Mean values of one representative experiment out of at least
three independent assays, were normalized relative to the CFU obtained in non
intoxicated infected cells.
FIG. 3. Uptake of B. abortus by HeLa cells treated with different bacterial toxins. A,
gentamicin survival assay of cells treated with different clostridial toxins and B,
gentamicin survival assay of cells treated with CNF. Mean values of one representative
experiment out of at least three independent assays, were normalized relative to the
CFU obtained in non-intoxicated infected cells.
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Brucella abortus internalization in HeLa cells 30
FIG. 4. The effect on B. abortus uptake in TcdB or CNF intoxicated HeLa cells
occurs before cytopathic effect is evident. A, gentamicin survival assay using TcdB-
1470 or TcdB-10463 treated HeLa cells at different time intervals. B, gentamicin
survival assay using CNF intoxicated HeLa cells at different time periods. The arrow
indicates the first time that cytopathic effect is observed. Bacteria were incubated with
cells after toxin treatment at each time point.
FIG. 5. Adhesion of virulent B. abortus to HeLa cells is not affected by TcdB-10463,
but is promoted in CNF intoxicated HeLa cells. A, HeLa cells were intoxicated with
TcdB-10463 for 15 or 40 min or with CNF for 2 h, infected with B. abortus for 30 min
and then extracellular (black bars) and intracellular bacteria (white bars) were counted
by double immunofluorescence analysis. Graph a, total number and proportion of
intracellular/extracellular bacteria/cell in TcdB-10463 intoxicated and non-intoxicated
HeLa monolayers. Graph b, total number and proportion of intracellular/extracellular
bacteria/cell in CNF intoxicated and non-intoxicated cells for both virulent B. abortus
2308 or non-pathogenic 2.13 strain. Graph c, number of cells with associated bacteria in
TcdB-10463 intoxicated and non intoxicated HeLa cells. Graph d, number of cells with
associated bacteria in CNF treated and non treated HeLa cells. Counts of intracellular
and extracellular bacteria were performed in at least 100 infected cells and expressed as
a mean of bacteria/cell obtained from one representative experiment out of three
independent assays. The percentage of cells with associated bacteria is expressed as the
mean of cells with bound bacteria in five different 40 × fields. The results presented are
from one experiment out of at least two independent assays. B, HeLa cells were
intoxicated with TcdB-10463 for 40 min or with CNF for 2 h, infected with B. abortus
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Brucella abortus internalization in HeLa cells 31
for 30min and then processed for immunofluorescence. Row a, extracellular bacteria
immunolabeled with FITC-conjugated anti-Brucella antibody. Row b, bacterial toxin
cytopathic effect showing spikes in TcdB-10463 treated cells (black arrows) and ruffles
(black arrow) in CNF intoxicated cells, as revealed by phase contrast microscopy. Row
c, superimposed images showing B. abortus attached to spikes of TcdB-10463 treated
cells (white arrows), or several bacteria bound to CNF treated cells (white arrows)
displaying membrane ruffles. Bacteria lying between the boundaries of cell to cell
contacts (white arrow) are shown in the control central column.
FIG. 6. B. abortus internalization is enhanced in HeLa cells expressing dominant
positive mutants of small GTPases. A, HeLa cells were microinjected with a plasmid
encoding the fusion protein Myc-RhoAV14 and infected with B. abortus for 30 min.
Cells were then fixed, permeabilized and processed for double immunofluorescence.
Frame a, microinjected cells had an altered morphology and were evident after
immunolabelling using a monoclonal anti-Myc antibody and a TRITC-conjugated anti-
mouse antibody. Frame b, immunolabelled bacteria using a FITC-conjugated anti-
Brucella antibody. Frame c, merged frames a and b demonstrate co-localization of
transformed cells with Brucella. Similar results were obtained when HeLa cells were
microinjected with plasmids encoding the fusion proteins Myc-Rac1V12 or Myc-
Cdc42V12 (not shown). B, number of bacteria per cell and proportion of cells with
intracellular bacteria in cells expressing dominant positive mutants of small GTPases.
Graph a, mean number of intracellular bacteria/cell found in at least 150 microinjected
cells. Graph b, percentage of cells expressing different dominant positive mutants with
intracellular bacteria. The results presented are from one experiment out of at least two
independent assays.
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Brucella abortus internalization in HeLa cells 32
FIG. 7. Expression of dominant negative mutants of small GTPases in HeLa cells
decreases B. abortus internalization. HeLa cells were transfected with plasmids
encoding the fusion proteins Myc-RhoAN19, Myc-Rac1N17 or Myc-Cdc42N17 and
infected with the virulent strain B. abortus 2308. The gentamicin survival assay was
then performed. Mean values are normalized relative to the CFU obtained in non-
transfected cells. The results presented are from one experiment of at least two
independent assays.
FIG. 8. Virulent B. abortus 2308 activates Cdc42 in HeLa cells. A, analysis of
activated Rho, Rac and Cdc42 using affinity precipitation at different times of infection
of HeLa cells with virulent strain B. abortus 2308 or the isogenic non-invasive mutant
strain 2.13. Samples were separated by SDS-PAGE, blotted and immunodetected with
either anti-Rho, Rac or Cdc42 antibodies. In the zero time point sample, tryptic soy
broth was added to cells. Samples from lysates were run in parallel by SDS-PAGE and
immunoblotted using specific anti-small GTPases antibodies to determine total amount
of each GTPase. Increased levels of Cdc42-GTP were detected after 30min infection
with the 2308 virulent strain. No differences in the quantities of Rho-GTP or Rac-GTP
were detected upon Brucella infection. B, quantification of Cdc42-GTP levels upon cell
interaction with virulent 2308 (open circles) and non-virulent 2.13 B. abortus strain
(closed circles) as compared to the negative control. One representative experiment out
of three different assays is presented.
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Brucella abortus internalization in HeLa cells 33
TABLE I. Comparative inhibition pattern of entry for Listeria and Salmonella.
Drug Target Effect on Listeria internalization Effect on Salmonella internalization
Colchicine Microtubules Inhibition in macrophages but not inHT-29 or Caco-2 enterocytes (99,100)
Not affected in CHO,HEp-2, MDCK, HT-29, Caco-2 and human epithelialcells (100-102)
Nocodazole Microtubules Inhibition in macrophages, non-proliferative HT-29 and IPI-2I cells(96,99)
Not affected in HeLa, MDCK and human epithelial cells(102-104)
2,3 Butanedione monoxime Actin-myosin interaction ND1 ND
Cytochalasin D Actin filaments 1 to 33% internalization in HeLa cells;inhibition in endothelial, Caco-2 andHT-29 cells; inhibition in HEp-2 cells(96,100,105-109)
Inhibition in HeLa, MDCK, CHO, HEp-2, Caco-2 andepithelial cells. Increased internalization in HT-29 andCaco-2 cells (100-104,110)
Tyrphostin Tyrosine protein kinases 10-100 fold inhibition in epithelialintestinal cell lines (111)
Not affected in HeLa cells (112)
Genistein Tyrosine protein kinases 10-100 fold inhibition in intestinal andepithelial cell lines; 47% internalizationin endothelial cells, inhibition inmacrophages, Caco-2 and HT-29 cells(106,108,109,111,113,114)
Not affected in HeLa, Henle 407 and A431 cells.Inhibition in Caco-2 and Ht-29 enterocytes (40,114)
PDO98059 Mitogen activated proteinkinases
25% internalization in HeLa cells (108) Not affected in HeLa cells or macrophages (55,108)
Wortmannin Phosphatidylinositol 3-kinase
25% internalization in HeLa cells, 1-2% internalization in Vero cells(43,108)
Mild inhibition in Vero cells. Inhibition of phagocytosis(43,55)
1 No data
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C
B D
A
Fig. 1 Guzmán-Verri et al.
34
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7nM 2,3 Butanedione monoxime
5µµµµg/ml Colchicine
10µµµµg/ml Nocodazole
1µµµµg/ml Cytochalasin D250µµµµM Tyrphostin
100µµµµM Genistein
50µµµµM PDO98059
50nM Wortmannin
CFU (%)
0 20 40 60 80 100
Fig. 2. Guzmán-Verri et al.
35
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0 20 40 60 80 100
CFU (%)
TcdB-10463
TcdB-1470
TcdA
TcsL-1522
Rac, Rho, Cdc42
Rac, Rap, R-Ras, Ral
Rac, Rho, Cdc42, Rap
Rac, Ras, Rap, R-Ras, Ral
Toxin Substrate
0 200 400 600 800 1000 1200
Rac, Rho, Cdc42
Toxin Substrate
CNF
CFU (%)
Control
A.
B.
Fig. 3. Guzmán-Verri et al.
36
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0
400
800
1200
0 4 8 12
0
25
50
0 50 100
minutes
% in
tern
aliz
ed
Bru
cell
a
CNF
hours
% in
tern
aliz
ed
Bru
cell
aA.
B.
TcdB-1470
TcdB-10463
Dose: 50ng/ml
Dose: 50ng/ml
Dose: 3ng/ml
Fig. 4 Guzmán-Verri et al.
37
4 8 50
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-2
0
2
4
-2
0
2
4
0
25
50
75
2308 2308+ 2308+ TcdB15’ TcdB40’
# ba
cter
ia/c
ell
2308 2308+ 2.13 2.13+ CNF CNF
# ba
cter
ia/ c
ell
% o
f ce
lls
wit
h a
ssoc
iate
d ba
cter
ia
2308 2308+ 2.13 2.13+ CNF CNF
A
a. b.
d.c.
0
10
20
% o
f ce
lls
wit
h a
ssoc
iate
d ba
cter
ia
2308 2308+ 2308+ TcdB15’ TcdB45’
38
Fig. 5. Guzmán-Verri et al.
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FIT
Can
ti-B
ruce
lla
Pha
seco
ntra
st
TcdB-10463 Control CNF
a.b
.c.
B
Fig. 5 Guzmán-Verri et al.
39
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0
2
4
6
8
10
# in
trac
ellu
lar
bac
teri
a/ce
ll
Control Rac+ Rho+ Cdc42+
0
20
40
60
80
% o
f ce
lls
wit
h in
trac
ellu
lar
bac
teri
a
Control Rac+ Rho+ Cdc42+
A.
B.
a b
a b
c
TRITC-anti-Myc FITC-anti-Brucella
Merged
40
Fig. 6. Guzmán-Verri et al.
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0 20 40 60 80
RhoAN19
Rac1N17
Cdc42N17
%CFU
Fig. 7. Guzmán-Verri et al.
41
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0
1
2
3
4
0 20 40 60 80
2308
2.13
Rho-GTP
Rac-GTP
Cdc42-GTP
Cdc42-GTP
Minutes
0 15 30 60
A.
B.
Minutes
Fol
d a
ctiv
atio
n
Fig. 8. Guzmán-Verri et al.
42
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MorenoLópez-Goñi, Monica Thelestam, Staffan Arvidson, Jean-Pierre Gorvel and Edgardo
Caterina Guzmán-Verri, Esteban Chaves-Olarte, Christoph von Eichel-Streiber, Ignacionon-professional phagocytes: direct activation of Cdc42
GTPases of the Rho subfamily are required for Brucella abortus internalization in
published online September 28, 2001J. Biol. Chem.
10.1074/jbc.M105606200Access the most updated version of this article at doi:
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